Our long-term goal is to elucidate the molecular mechanism of the cytoplasmic dynein-dynactin motor complex, and to define the molecular bases of dynein-related diseases in humans. Dynein is the primary vehicle for microtubule minus-end-directed transport in eukaryotic cells. The function and dysfunction of this vital motor and its regulatory proteins contribute to a broad set of cellular functions and human diseases. Despite increasing efforts to define dynein's functional properties, the molecular mechanisms that govern dynein's mechanochemistry remain poorly understood. This deficiency largely stems from dynein's structural complexity. Dynein belongs to the AAA+ class of ATP-hydrolyzing mechanoenzymes that assemble into ring- shaped structures, and therefore, possesses characteristically distinct structural features compared to the other two cytoskeletal motor protein families, kinesin and myosin. Dynein is also exceptionally large (~1.2 MDa) and structure-function studies on dynein have been limited by the availability of functional recombinant dynein. Adding to dynein's complexity, dynein associates with multiple accessory chains and the dynactin complex, all of which are essential for nearly every cellular function of dynein. Mutations in dynactin's largest subunit, p150glued, which contains dynactin's putative microtubule-binding domain, cause Perry syndrome and motor neuron degeneration in humans. Yet, the role of p150glued in dynein function remains unknown. In this grant, we seek to overcome these limitations by combining ultrasensitive single-molecule assays with protein engineering. We will use S. cerevisiae, the only source for recombinant full-length dynein and dynactin, to produce stable wildtype and mutant versions of both multiprotein complexes. Using these biochemical tools and multicolor single-molecule fluorescence and optical tweezers methods, we will resolve 1) how dynein's AAA+ motor domains are coordinated within dynein's mechanochemical cycle, 2) how dynactin modulates and regulates dynein function, and 3) how human p150glued mutations disrupt the function of the dynein-dynactin complex. This information will provide insight into cellular physiology and pathophysiology, and potentially identify targets within the dynein-dynactin complex for therapeutic interventions.